title: Pigment epithelium-derived factor (PEDF) prevents retinal cell death via PEDF Receptor (PEDF-R): identification of a functional ligand binding site.
abstract: The extracellular pigment epithelium-derived factor (PEDF) displays retina survival activity by interacting with receptor proteins on cell surfaces. We have previously reported that PEDF binds and stimulates PEDF receptor (PEDF-R), a transmembrane phospholipase. However, the PEDF binding site of PEDF-R and its involvement in survival activity have not been identified. The purpose of this work is to identify a biologically relevant ligand-binding site on PEDF-R. PEDF bound the PEDF-R ectodomain L4 (Leu(159)-Met(325)) with affinity similar to the full-length PEDF-R (Met(1)-Leu(504)). Binding assays using synthetic peptides spanning L4 showed that PEDF selectively bound E5b (Ile(193)-Leu(232)) and P1 (Thr(210)-Leu(249)) peptides. Recombinant C-terminal truncated PEDF-R4 (Met(1)-Leu(232)) and internally truncated PEDF-R and PEDF-R4 (ΔHis(203)-Leu(232)) retained phospholipase activity of the full-length PEDF-R. However, PEDF-R polypeptides without the His(203)-Leu(232) region lost the PEDF affinity that stimulated their enzymatic activity. Cell surface labeling showed that PEDF-R is present in the plasma membranes of retina cells. Using siRNA to selectively knock down PEDF-R in retina cells, we demonstrated that PEDF-R is essential for PEDF-mediated cell survival and antiapoptotic activities. Furthermore, preincubation of PEDF with P1 and E5b peptides blocked the PEDF·PEDF-R-mediated retina cell survival activity, implying that peptide binding to PEDF excluded ligand-receptor interactions on the cell surface. Our findings establish that PEDF-R is required for the survival and antiapoptotic effects of PEDF on retina cells and has determinants for PEDF binding within its L4 ectodomain that are critical for enzymatic stimulation.
authors.affiliation: Section of Protein Structure and Function, Laboratory of Retinal Cell and Molecular Biology, NEI, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: Preeti
authors.initials: P
authors.lastname: Subramanian
authors.forename: Silvia
authors.initials: S
authors.lastname: Locatelli-Hoops
authors.forename: Jason
authors.initials: J
authors.lastname: Kenealey
authors.forename: Jacqueline
authors.initials: J
authors.lastname: DesJardin
authors.forename: Luigi
authors.initials: L
authors.lastname: Notari
authors.forename: S Patricia
authors.initials: SP
authors.lastname: Becerra
mesh_terms: D000818:Animals; D001665:Binding Sites; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D005136:Eye Proteins / Q000378:metabolism* / Q000494:pharmacology; D006801:Humans; D008024:Ligands; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009414:Nerve Growth Factors / Q000378:metabolism* / Q000494:pharmacology; D010455:Peptides / Q000378:metabolism / Q000494:pharmacology; D010740:Phospholipases / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018013:Receptors, Neuropeptide / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012160:Retina / Q000166:cytology*; D015843:Serpins / Q000378:metabolism* / Q000494:pharmacology; D000099174:Pigment Epithelium-Derived Factor
chemical_list: D005136:Eye Proteins; D008024:Ligands; D050505:Mutant Proteins; D009414:Nerve Growth Factors; D010455:Peptides; D010740:Phospholipases; D018013:Receptors, Neuropeptide; D011993:Recombinant Fusion Proteins; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor; C400531:pigment epithelium-derived factor receptor
keywords: Cell Surface Receptor; Ligand-binding Protein; Receptors; Retina; Retinal Degeneration